486
Autoimmunity in Scleroderma
the immunization process in scleroderma
autoimmunity.
1.3
Detection of Scleroderma Autoantibodies
and Epitope Analysis
Detection of the scleroderma autoanti-
bodies depends on the assay systems.
For example, Kumar et al. reported that
the anti-topo I sera that were positive
on the immunoblot were only 71 to
86% positive in the immunodiffusion test.
Similar results were obtained by Hilde-
brandt; anti-topo I IgG were 88% posi-
tive by ELISA with the puri±ed topo I
from calf thymus, in contrast to being
100% positive by immunoblotting. In ad-
dition, Maul et al. reported that a few
anti-topo I–negative sera were positive
by double diffusion with the recombi-
nant protein blotted on the membrane.
Sensitivity of ACA detection was also
compared by Hildebrandt et al. among
ELISA, immunoblotting, and IIF. IgG-
ACA was detected to be 85% by ELISA
with cloned C-terminal CENP-B antigen,
95% by immunoblotting with HeLa chro-
mosomal proteins, and 100% by IIF with
HEp-2 cells. These results indicate that
among the above detection systems, im-
munoblotting seems to display a compa-
rable sensitivity for the detection of these
autoantibodies, even if these antigens have
some conformational epitope (described
below).
Epitope mapping studies have been
performed more extensively with B-cell
than
with
T-cell
studies
because
au-
toantibodies (B cells) are easy to han-
dle. Various approaches have been used
for the studies; for example, deletion-
type immunoblotting experiments with
recombinant fragments of the autoanti-
gen and ELISA with synthesized peptides
covering the whole sequence of the au-
toantigen. The former method somehow
detects conformational epitopes, consist-
ing of amino acid residues from distant
regions in the sequence of a protein that
are spatially juxtaposed upon, while the
latter method is suitable for the detection
of linear epitopes in the primary structure
of a protein. Since the epitope regions are
renatured to some extent during protein
transfer and while blocking the mem-
brane, the immunoblot analysis can reveal
the linear and conformationally reversible
epitopes.
The major autoantigens associated with
scleroderma are shown in Table 1: topo
I, CENP-A, CENP-B, CENP-C, 75 kDa
and 100 kDa antigens of PM/Scl, and
±brillarin. Epitope analyses of the epi-
tope structure for these autoantigens at
the molecular level would provide a sig-
ni±cant insight into the interactions be-
tween the antigen and the autoantibodies.
What are the trigger components lead-
ing to autoimmune response? (pathogens?
or the antigens themselves?) Are there
some
characteristic
features
of
struc-
tures targeted by autoantibodies? How
are autoantibodies related to the disease
process? We focus on the epitope struc-
ture of scleroderma autoantigens, mainly
describing the analysis of centromere
antigens.
2
Centromere Antigens
2.1
Autoimmunity against Centromere
Antigens
The attachment site of spindle micro-
tubules on mitotic chromosomes is called
the
centromere
. The centromere has a
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